Chemogenetic stimulation of the Gi pathway in astrocytes suppresses neuroinflammation

Abstract Engineered G protein‐coupled receptors (GPCRs) are commonly used in chemogenetics as designer receptors exclusively activated by designer drugs (DREADDs). Although several GPCRs have been studied in astrocytes using a chemogenetic approach, the functional role of the astrocytic Gi pathway is not clear, as the literature is conflicting depending on the brain regions or behaviors investigated. In this study, we evaluated the role of the astrocytic Gi pathway in neuroinflammation using a Gi‐coupled DREADD (hM4Di). Gi‐DREADD was expressed in hippocampal astrocytes of a lipopolysaccharide (LPS)‐induced neuroinflammation mouse model using adeno‐associated viruses. We found that astrocyte Gi‐DREADD stimulation using clozapine N‐oxide (CNO) inhibits neuroinflammation, as characterized by decreased levels of proinflammatory cytokines, glial activation, and cognitive impairment in mice. Subsequent experiments using primary astrocyte cultures revealed that Gi‐DREADD stimulation significantly downregulated LPS‐induced expression of Nos2 mRNA and nitric oxide production. Similarly, in vitro calcium imaging showed that activation of the astrocytic Gi pathway attenuated intracellular calcium transients triggered by LPS treatment, suggesting a positive correlation between enhanced calcium transients and the inflammatory phenotype of astrocytes observed in the inflamed brain. Taken together, our results indicate that the astrocytic Gi pathway plays an inhibitory role in neuroinflammation, providing an opportunity to identify potential cellular and molecular targets to control neuroinflammation.


| INTRODUC TI ON
Designer receptors exclusively activated by designer drugs (DREADDs) are genetically modified G-protein-coupled receptors (GPCRs). DREADDs are used in chemogenetic approaches, which allow researchers to remotely control cellular activity via modulation of GPCR (G i , G q , or G s )-signaling pathways with the application of selective ligands, such as clozapine N-oxide (CNO). 1 This strategy has commonly been used to regulate the activity of various types of neurons to study brain functions and behaviors. Recent studies have also used this technique to study glial cells, including astrocytes, 2,3 the most abundant glial cell type in the central nervous system (CNS), exhibiting notable heterogeneity in their morphology and function. 4 Among the GPCRs in astrocytes studied using DREADDs, there are inconsistencies reported in the functional role of G i signaling, which requires further exploration.
The functional role of the G i signaling pathway in astrocytes has been investigated in several studies using chemogenetic approaches. A study by Nam et al. revealed that chemogenetic activation of astrocyte-specific G i -DREADD hM4Di enhanced synaptic transmission and plasticity of Schaffer collaterals in the hippocampus, thereby inducing the formation of contextual memory for conditioned place preference. 5 Conversely, another recent study demonstrated that activation of G i -coupled designer receptor hM4Di in hippocampal cornu ammonis (CA1) astrocytes during learning impairs remote, but not recent memory recall, and decreases the activity of CA1 neurons projecting to the anterior cingulate cortex (ACC) during memory retrieval. 6 Similarly, another study found that activation of astroglial G i signaling in the hippocampus was sufficient to protect against the development of stress-enhanced fear learning, post-traumatic stress disorder-like behavior. 7 Moreover, it has been demonstrated that striatal astrocyte G i pathway activation corrects behavioral phenotypes in a Huntington's disease mouse model. 8 However, the same research group previously reported that activation of an astrocyte-specific G i pathway in the striatum produced inattentive hyperactivity in mice under physiological conditions. 9 Therefore, given the multiple contradicting results, the functional role of the astrocytic G i pathway remains unclarified in both healthy and disease states. It is well accepted that in disease conditions astrocytes can undergo morphological and functional remodeling into "reactive astrocyte," called "astrogliosis," where normal homeostatic mechanisms are lost and proinflammatory responses occur at higher levels, contributing to neuroinflammation and associated diseases. 10,11 Astrocyte-mediated neuroinflammation is associated with neurodegenerative and metabolic diseases, such as Alzheimer's disease (AD), 12 Parkinson's disease (PD), 13 traumatic brain injury (TBI), 14 multiple sclerosis (MS), 15 diabetes, and obesity. 16 However, to the best of our knowledge, the role of the astrocytic G i pathway in neuroinflammation has not been yet studied.
To explore the role of astrocytic G i pathway in neuroinflammation, we used the designer receptor hM4Di to manipulate the G i pathway in these cells in a lipopolysaccharide (LPS)-induced neuroinflammation model. We found that chemogenetic activation of astrocytic G i signaling in the hippocampus attenuates LPSinduced neuroinflammation, as evidenced by decreased levels of inflammatory mediators, gliosis, and cognitive impairment in mice.
In vitro studies using cultured astrocytes revealed that G i activation in astrocytes reduced LPS-induced expression of Nos2 mRNA, nitric oxide (NO) production, and intracellular calcium (Ca 2+ ) levels.
These findings provide evidence for the important role of astrocytic G i activation and downstream signaling pathways in mitigating neuroinflammation.
Only male mice were used in this study. All animal experiments were
After injection, the needle tip was held in place for 10 min before retraction to prevent leakage, and then removed. Immediate postoperative care was provided, and the animals were allowed to recover for 14 days before the experiment, to ensure high levels of transgene expression. Prior to behavioral experiments following viral gene transfer, the expression of relevant proteins within the CA1 region was confirmed via fluorescence.

| CNO administration
CNO (Tocris Bioscience, Catalog number: 4936) was dissolved in DMSO and then diluted in 0.9% saline to yield a final DMSO concentration of 0.5%. The saline solution used for control injections also consisted of 0.5% DMSO. Before conducting the behavioral assays, 1 or 3 mg/kg CNO was intraperitoneally (i.p.) injected at 8-h intervals for 2 days. Despite the short CNO half-life in mouse plasma (<2 h), 17 acute treatment of DREADD-expressing experimental animals usually have much longer biological effects (6-10 h). [17][18][19] To investigate the effect of astrocyte chronic activation, we injected CNO (1 or 3 mg/kg, i.p.) into mice at 8-h intervals. We chose this 8-h duration based on a previous report. 19 For in vitro studies, primary astrocytes expressing hM3Dq-or hM4Di-mCherry were treated with CNO (10 μM).

| Intracerebroventricular injection of LPS
Under isoflurane anesthesia, mice were mounted onto a stereotaxic frame. Two guide cannulas were surgically implanted bilaterally 0.5 mm above the lateral ventricle of the brain. The

| Immunohistochemistry
Animals were anesthetized using diethyl ether, and transcardially

| Reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was extracted from hippocampal tissues and cells using the QIAzol reagent (QIAGEN) according to the manufacturer's protocol. For conventional RT-PCR, reverse transcription was conducted using Superscript II (Invitrogen) and oligo(dT) primers. PCR amplification using specific primer sets was carried out at an anneal-

| Passive avoidance test
This test began with training, in which a mouse was placed in a light chamber; when the mouse crossed over to the dark chamber, it received a mild electric shock on the foot (0.25 mA for 1 s). The initial latency to enter the dark (shock) compartment was used as the baseline measure. During the probe trials, 24 h after training, the mouse was again placed in the light compartment, and the latency to return to the dark compartment was measured as an index of passive fear avoidance. The passive avoidance test method used for cognitive behavior assessment has a 200 s maximum latency, as observed with most of the vehicle and CNO-treated animals. Thus, it is not suited for cognitive enhancement observation, which is similar to previously published studies. 21 Future studies will need to address this issue using other methods such as novel object recognition task, Barnes maze, or Morris water maze tests. 22,23

| Primary astrocyte cultures and virus infection
The brains of 3-day-old mice were homogenized and mechanically disrupted using a nylon mesh. The obtained mixed glial cells were seeded in culture flasks, and cultured at 37℃ in a 5% CO 2 incubator in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ ml streptomycin. Culture media were changed initially after 5 days, and then changed every 3 days. After 14 days of culture, primary astrocytes were obtained from mixed glial cells using a mechanical shaker (200 rpm for 12 h). Then, primary astrocytes in 12-well plates were infected with hM3Dq-or hM4Di-mCherry virus. After 7 days of virus infection, the cells were used for experiments.

| Nitrite quantification
Astrocyte cultures were treated with stimuli in 96-well plates, and then NO 2 − in culture media was measured in order to assess NO production levels by the Griess reaction as described previously. 24 Sample aliquots of 50 μl were mixed with 50 μl of Griess reagent (1% sulfanilamide, 0.1% naphthylmethyl diamine dihydrochloride, 2% phosphoric acid) in 96-well plates, and then incubated at 25℃ for 10 min. The absorbance at 540 nm was measured using a microplate reader (Anthos Labtec Instruments). NaNO 2 was used as a standard to calculate NO 2 concentration.

| Intracellular Ca 2+ measurement
To measure intracellular Ca 2+ in cultured astrocytes, we grew primary We recorded the confocal images every 1.5 s for 460 s. The illumination intensity was limited to 0.5%-0.7% of the laser output.
To measure the effect of CNO alone on intracellular Ca 2+ levels in hM4Di-expressing astrocytes, we recorded green fluorescence images every 0.5 s for 10 s (baseline) using a Lionheart FX Automated Microscope with a 10× phase lens. We then injected 10 μl of 10 μM

CNO into the cells using the injector of the Lionheart FX Automate
Microscope. We recorded images every 0.5 s for 170 s.

| Statistical analysis
All data are presented as means ± SEM (in vivo data) or means ± SD

| Chemogenetic stimulation of G i signaling in hippocampal astrocytes ameliorates LPS-induced cognitive impairment in mice
Accumulating evidence suggests that neuroinflammation is associated with impaired cognitive function in diverse neuropathological conditions. 26 Figure 3A and Figure S2). The chemogenetic stimulation of astrocytic G i signaling (CNO at 3 mg/kg) increased the latency to escape in LPS-injected animals 24 h after a foot shock, indicating that astrocytic G i signaling ameliorates LPS-induced cognitive deficit ( Figure 3B, right).
The latency during the training trial did not differ among the experimental and control groups, indicating that all the mice had similar responses to the testing environment ( Figure 3B, left). The escape latency was not affected by CNO treatment at a low dose (1 mg/kg) ( Figure S2C). These results imply that strong activation of G i signaling in hippocampal astrocytes has an inhibitory effect on LPS-induced neuroinflammation and on subsequent cognitive impairment in mice.

| Chemogenetic stimulation of G i signaling in cultured astrocytes attenuates LPS-induced nitric oxide production
Since nitric oxide (NO) production has been used as an indicator of inflammation in astrocytes, 28 we investigated the effect of astrocytic G i signaling activation on LPS-induced NO production. Primary astrocytes were infected with a AAV5-hGFAP-hM4Di-mCherry virus construct and stimulated with LPS for 24 h ( Figure 4A). Subsequently, the accumulated nitrite in the culture media was estimated using Griess reaction as an index for NO synthesis. Exposure of primary astrocyte cultures to LPS markedly increased the levels of nitrite in the culture media ( Figure 4B).
However, hM4Di activation by CNO treatment in primary astrocytes significantly decreased LPS-induced NO production.
We also used a positive control chemogenetic stimulation of the G q -signaling pathway, by infecting astrocyte cultures with a AAV5-hGFAP-hM3Dq-mCherry virus construct. As expected, the activation of the astrocytic G q -signaling pathway using CNO increased the levels of nitrite in the media. Next, to investigate whether the inhibitory effect of astrocytic G i signaling on LPSinduced NO production was mediated by iNOS suppression, we performed RT-PCR analysis and found that activation of G i  Figure 4C). These findings support the inhibitory role of astrocytic G i signaling in neuroinflammation.

| Chemogenetic stimulation of G i signaling in cultured astrocytes attenuates LPS-induced intracellular Ca 2+ transients
Ca 2+ signals in astrocytes change both acutely and chronically in response to brain insults, such as injury and inflammation. 29 However, it is unclear how G i signaling in astrocytes affects Ca 2+ signals during neuroinflammation. To test this, we performed Ca 2+ imaging in cultured astrocytes expressing either hM4Di, hM3Dq (a positive control), or without infection (a negative control), in which cultured astrocytes were loaded with Fluo-4-AM as shown in Figure 5A. The primary astrocytes expressing hM4Di were imaged before and after application of PBS, LPS, and CNO (10 μM) ( Figure 5B,C). The data revealed that CNO application following LPS treatment reduced LPSinduced intracellular Ca 2+ levels in hM4Di-expressing astrocytes ( Figure 5B). The application of CNO prior to LPS also prevented LPS-induced upregulation of intracellular Ca 2+ levels in hM4Diexpressing astrocytes ( Figure 5C). However, hM3Dq-expressing astrocytes showed an increase in intracellular Ca 2+ levels following Immunofluorescence staining and image analysis were performed. Astrocytes were immunohistochemically labeled with GFAP (white), and microglia were labeled with Iba-1 (red or green). Quantitative analysis of GFAP-positive astrocytes and Iba-1-positive microglia as well as relative GFAP and Iba-1 immunoreactivity (IR) intensity in the hippocampus are presented in adjacent graphs. Scale bar, 200 μm. Results are expressed as means ± SEM (n = 4). *p < .05 between the indicated groups (one-way ANOVA with Bonferroni's post hoc test). eYFP, AAV5-hGFAP-eYFP; hM4Di-mCherry, AAV5-hGFAP-hM4Di; IR, immunoreactivity; ns, not significant CNO application ( Figure 5D). As shown in Figure 5C, CNO application acutely increased intracellular Ca 2+ levels in hM4Di-expressing astrocytes. Thus, to better characterize the effect of intracellular Ca 2+ levels in hM4Di-expressing astrocytes, we measured intracellular Ca 2+ levels in hM4Di-expressing astrocytes treated with CNO alone. Treatment with CNO alone increased intracellular Ca 2+ levels in hM4Di-expressing astrocytes ( Figure S3). Similarly, chemogenetic activation of either the G q or G i pathway increased intracellular Ca 2+ in astrocytes. 9,30-32 However, whereas G q pathway activation resulted in a long-lasting increase of Ca 2+ activity (Figure 5D), the G i pathway-induced intracellular Ca 2+ transient wanes in time ( Figure   S3). Collectively, these findings suggest that the alteration in intracellular Ca 2+ levels following G i or G q pathway activation in astrocytes may reflect the features of reactive astrocytes associated with neuroinflammation.

| DISCUSS ION
Our findings demonstrate that chemogenetic stimulation of the astrocytic G i signaling pathway in the hippocampus plays an inhibitory role in neuroinflammation and subsequent cognitive decline in mice.
Stimulation of G i signaling was sufficient to inhibit proinflammatory activation of astrocytes, which correlated with intracellular Ca 2+ transients.
In this study, we have shown that long-term hM4Di activation ameliorates LPS-induced production of proinflammatory mediators and cognitive impairment, suggesting that G i signaling could lead to suppression of inflammatory activation of astrocytes and concurrent neuroinflammatory changes. The role of astrocytic GPCR signaling in neuroinflammation has been previously reported. 33 The astrocyte dopamine receptor (DRD)-2 coupled to G i 34,35 has been found to decrease in the aging brain, 36 implying a potential involvement of DRD2 in aging-related neuroinflammation. It has been reported that DRD2-deficient astrocytes produce higher levels of proinflammatory molecules. 37 This effect is mediated through inhibition of αB-crystallin signaling, a small heat-shock protein known to negatively regulate the production of proinflammatory mediators and to exhibit neuroprotective effects. Intriguingly, DRD2-deficient astrocytes also display robust upregulation of GFAP expression with a reactive morphology in the substantia nigra and the striatum of aged mice, 33 suggesting a possible link between astrocytic G i signaling and age-related neuroinflammation and subsequent behavioral impairment.
Conversely, dopaminergic signaling triggered by the stimulation of G i -coupled DRD3 promotes a proinflammatory phenotype in astrocytes. 38 Moreover, G i -coupled P2Y12R and P2Y14R signaling has also been reported to be involved in proinflammatory activation of astrocytes and immune cells. [39][40][41][42][43][44][45] In this study, chemogenetic stimulation of G i signaling in pri- activation of G i signaling in astrocytes suppresses cAMP. 54  receptors. 57 Reactive astrocytes show elevated Ca 2+ levels upon various inflammatory stimuli. 58 It has been reported that the upregulation of astrocytic Ca 2+ is also essential for GFAP upregulation (a marker for reactive astrocytes) in diverse neuropathologies, including AD, 59 Alexander disease, 60 photothrombosis, 59 and traumatic brain injury, 61 whereas abrogation of aberrant Ca 2+ signals (via IP 3 receptor KO, etc.) strongly suppresses GFAP upregulation and subsequent inflammatory phenotypes.
Astrocytes express toll-like receptor type 4 (TLR4), which belongs to the TLR family in the vertebrate immune system and specifically recognizes LPS. 62 On astrocytes, LPS decreases the expression of proteins such as gap junction proteins 63 and increases the expression of others such as GFAP, s100β, IL-1, and TNFα. 6  blocked the Ca 2+ -induced peak. 69 The authors have found that LPS-induced Ca 2+ transients oscillate, Na + /K + -ATPase is downregulated, and the actin filaments are disorganized. 69 The Na + / K + -ATPase is an energy-transducing pump, and its expression decreased with time. Modulating this pump's activity affects intracellular Na + concentration, which in turn changes intracellular Ca 2+ concentration via Na + -Ca 2+ exchanges. 70 Therefore, these data implicate L-type Ca 2+ channels in the LPS-induced activation of astrocytes. 71 However, identifying the molecular pathways involved in L-type VOCCs modulation by LPS requires further studies. eYFP, AAV5-hGFAP-eYFP; hM3Dq-mCherry, AAV5-hM3Dq-mCherry; hM4Di-mCherry, AAV5-hM4Di-mCherry In conclusion, our findings suggest that the astrocytic G i pathway plays an inhibitory role in neuroinflammation via downregulation of proinflammatory mediators, NO production, and intracellular Ca 2+ levels. These results provide opportunities to identify potential cellular and molecular targets for the control of neuroinflammation.

D I SCLOS U R E
The authors declare no conflicts of interest in regards to this manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data pertinent to this work are contained within this manuscript or available upon request. For requests, please contact: Kyoungho Suk, Kyungpook National University, ksuk@knu.ac.kr.